Effectiveness of CRISPR-Cas9 using pools of synthetic crRNAs in high-content analysis screening experiments

Introduction Gene knockout using the CRISPR-Cas9 system has emerged as a powerful technology for loss-of-function screening. Although screening using pooled lentiviral sgRNA libraries is a powerful way of discovering gene function1,2, arrayed screening expands the types of phenotypic readouts from simple population enrichment or depletion to complex multiparametric high-content imaging and morphological assays. Chemical synthesis of guide RNAs for CRISPR-Cas9 gene editing allows for accurate and rapid production of CRISPR libraries and enables screening in an arrayed, one-gene-per-well fashion. Several high-throughput arrayed screens using synthetic crRNA as the CRISPR guide RNA have been published3-7. These screens were performed using a single crRNA per well with multiple unique crRNAs per gene for more data points, and therefore increased confidence in hit identification.